More than 36 million people worldwide are living with HIV-1 infection as of 2017, with HIV/AIDS causing ~1 million deaths per year. HIV-1 establishes a life-long, persistent infection. There are no therapies yet capable of permanently suppressing viral gene expression in the context of acute infection or latency rebound. This project?s long-term goal is to elucidate the cellular mechanisms that underpin HIV-1 RNA subcellular trafficking, translation, and genome packaging toward the development of therapies to selectively abrogate these stages in vivo. In the current funding period we elucidated cell-intrinsic barriers to HIV-1 genome nuclear export in cells derived from mice and other rodents. We also developed cutting- edge, quantitative imaging strategies for studying HIV-1 viral RNA (vRNA) trafficking and virus particle assembly dynamics in human cells. Collectively, these studies revealed that cooperative interactions between discrete cis-acting viral RNA structural elements and defined RNA binding proteins program vRNAs for striking transport behaviors both in the nucleus and cytoplasm. For example, we found that HIV- 1?s Rev response element (RRE), regulated by the viral Rev protein and cellular XPO1 nuclear export receptor, dictates a previously unanticipated 3-step vRNA transport pathway characterized by transient subnuclear compartmentalization events, ?burst? nuclear export kinetics, and diffusion to peripheral sites of translation and genome packaging in the cytoplasm. Herein we test the overarching hypothesis that HIV-1 is adapted to exploit XPO1-mediated ?burst? export in order to ensure rapid, non-linear increases to viral late stage gene expression and to promote the efficient delivery of viral genomes to virion assembly sites at the cell periphery. The goal of Specific Aim 1 is to define the nuclear membrane events that underpin XPO1-directed ?burst? vRNA nuclear export using advanced high-resolution imaging modalities. Specific Aim 2 applies a comparative visual and biochemical approach to define conserved features of XPO1-linked vRNA export modules in the context of broad-spectrum antiviral targeting. Specific Aim 3 uses cell-based assays and new HIV-1 reporter viruses to study the links between ?burst? export at the nucleus and cytoplasmic events including Gag/Gag-Pol translation, genome packaging, and virus particle assembly. Collectively, these detailed studies are intended to expose new cell biology, deliver innovative tools for studying viruses, and identify novel virus-host interactions relevant to the development of therapies to suppress HIV-1 virion production in vivo.